The long-range objective is to understand the mechanisms involved in coupled exocytosis and endocytosis in neuroendocrine cells. In these cells, exocytosis of dense-core vesicles (DCVs) is used to secrete hormones and biogenic amines that regulate most of man's internal functions, and endocytosis recycles DCV membranes for reutilization. Recent insight indicates that coupling of these processes controls secretion quantitatively, providing for specific signaling between organs through regulating signaling strength. In adrenal medulla, coupled exocytosis and endocytosis controls secretion of catecholamines, adrenalin and noradrenalin. Clarifying how coupling works is essential for defining physiological events that are potential targets in endocrine pathologies involving hypertension and systemic stress. Proposed studies focus on the roles of Secretory Carrier Membrane Proteins (SCAMPs) in exo-/endocytic coupling in adrenal medulla- derived pheochromocytoma (PC12) cells and mouse adrenal chromaffin cells. One isoform, SCAMP2, interacts with three proteins that function in exocytosis - small G protein Arf6, phospolipase D1 (PLD1), and phosphatidyl inositol 4-phosphate 5-kinase (PIP5K) - and also participates in opening and dilating fusion pores in DCV exocytosis. Other SCAMPs bind complexin (SCAMP1) and dynamin (SCAMPs 1 &5) - interactions which are thought to support DCV exocytosis and endocytosis. These findings have led to the hypothesis that SCAMPs organize and couple opening and closing steps of exo-/endocytosis. Four aims will evaluate aspects of this hypothesis. 1) SCAMP interactions with Arf6, PLD1, PIP5K, and complexin involved in exocytosis will be mapped and tested for effects on exocytosis mainly using amperometry. 2) Ability of peptides and full-length SCAMPs to sequester phosphoinositide PIP2, required for exo-/endocytosis, will be evaluated using biophysical assays and fluorescence microscopy of cells deficient in SCAMPs or expressing SCAMP mutants defective in lipid sequestration. 3) SCAMP function in endocytosis, particularly involving dynamin interaction, will be examined mainly by fluorescence microscopy to analyze dynamin recruitment and DCV membrane recovery where dynamin-SCAMP interactions are perturbed. 4) Exocytosis and exo- endocytic coupling will be analyzed in chromaffin cells lacking SCAMP1 using amperometry, tracer uptake, and electron microscopy to analyze defects thought to be related to chromaffin cell physiology.